We have initiated studies of Myo1G in lymphocytes, because of our finding that: 1) Myo1G is highly enriched in lymphocyte membrane / microvilli;2) Myo1G is previously uncharacterized;and 3) Myo1G is expected to be involved in regulating cortical organization (based on other Myo isoforms in eukaryotes). We have generated a structure based sequence alignment, and generated antibodies and GFP fusion proteins to show that Myo1G is present exclusively at the plasma membrane in lymphocytes. The PH domain in class I myosins is divergent and may represent an ancestral PH domain, since our analysis identified for the first time a homologous PH domain in bacteria. Overexpression studies and mutational analysis of conserved basic residues implicated in ligand binding showed that Myo1G membrane localization is in part due to the PH domain that, which we find is present in the tail of all class I myosins (Myo1A-H). Using a set of deletion and mutation constructs we demonstrated that the PH domain is necessary but not sufficient for membrane localization of Myo1G and have characterized the additional requirements. We have modeled the PH domain and mutated multiple basic residues to identify the critical residues for membrane localization. Analysis of lipid binding will be undertaken upon completion of appropriate constructs and recombinant proteins. Perturbations of membrane lipids in intact cells reveals different requirements for Myo1G from the previously described requirements for Myo1C. . In summary, these studies shown that there is a conserved PH domain in all human class I myosins, and this domain is important for normal localization, however the specificity for interaction with lipids at the plasma membrane differs between them. In order to understand the broader biological function of Myo1G, we have generated a Myo1G conditional knockout mouse and are characterizing functional alterations. We have also initiated studies of NHERF1, a PDZ-containing adaptor molecule important in regulating molecular localization and function in multiple cells types. An additional rationale for study is recent evidence implicating NHERF1 in cancer. But the function of NHERF1 in the immune system is largely unstudied. The importance of ERM binding to NHERF1 is regulating localization at the membrane has been described but is not well understood. We have therefore undertaken structure function analysis to better characterize this process, especially with regard to the role of regulation by membrane lipid and autoinhibition. In addition, we have initiated a collaboration to study knockout mice, and are back-crossing the mouse strain to Bl6 to improve their usefulness for immunologic analysis. Initial results indicate that knockout lymphocytes have alterations in cell spreading and migration.